Mapping local nucleotide flexibility by selective acylation of 2 '-amine substituted RNA

2'-Amine substitutions in single-stranded oligoribonucleotides react mon ra pidly with activated esters than 2'-amine positions in mismatched or duplex RNA substrates. Reactivity does not reflect static solvent accessibility o r electrostatics. We infer that acylation of 2'-amine substituted RNA is se nsitive to local nucleotide flexibility. Selective acylation was used to ma p the structure and magnesium ion dependent conformational changes in tRNA( Asp) transcripts containing single 2'-amine substitutions per transcript. U nder denaturing conditions, all T-amine substituted RNA positions show simi lar reactivity. When tRNA(Asp) transcripts are refolded under strongly nati ve conditions (10 mM Mg2+, 100 mM NaCl), positions involved in base pairing and known tertiary interactions, including base triples and loop-loop inte ractions, are protected from modification. In the absence of magnesium ion the acceptor. T- and anticodon stems form stable helices as judged by their low relative 2'-amine reactivity. In contrast, the D-stem and most tertiar y interactions require greater than 1 mM MgCl2 for stable folding. These re sults emphasize an interdependence between formation of the D-stem helix an d tertiary structure folding for yeast rRNA(Asp) transcripts. This chemical approach fur mapping local RNA flexibility yields results consistent with prior biophysical and biochemical studies emphasizing its utility for mappi ng local nucleotide environments on small quantities of RNA molecules of an y size.